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The generation of mast cells for in vitro studies comes from a variety of sources including mast cell lines (MC/9) (McCurdy et al., 2001), bone marrow-derived mast cells (BMMCs) (Supajatura et al., 2001), skin-derived mast cells (FSMCs) (Matsushima et al., 2004), peritoneal-derived mast cells (PMCs) (Hochdorfer et al., 2011) and peritoneal cell-derived cultured mast cells (PCMCs) (Vukman et al., 2012). BMMCs are generally used for in vitro studies because of the high yield of mast cells generated and also because they can be generated from knockout and transgenic mice making this a good source to examine specific factors important for mast cell function. Due to the large yield of cells generated they are the cells of choice for reconstitution studies in mast cell knockout mice (Sur et al., 2007). Furthermore, they are more responsive to both allergic and non-allergic stimuli when compared to mast cell lines. The major disadvantage of BMMCs is that they are not fully matured when compared to mast cells generated or obtained from other sources. For example, compared to PCMCs [see the protocol “Isolation and Culture of Peritoneal Cell-derived Mast Cells” (Vukman et al., 2014)], BMMCs express a restricted range of TLRs and cytokines when stimulated with TLR ligands (Mrabet-Dahbi et al., 2009). The different sources of mast cells can display phenotypical and functional differences and therefore it is important that when designing an experiment the correct cellular source is obtained. Here, we describe a protocol for the isolation and culture of murine mast cells from mouse bone marrow.

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Isolation and Culture of Bone Marrow-derived Mast Cells

Immunology > Immune cell isolation > Mast cell
Authors: Krisztina V. Vukman
Krisztina V. VukmanAffiliation: Biotechnology Department, Dublin City University, Dublin, Ireland
For correspondence: sztitussz@gmail.com
Bio-protocol author page: a1175
Martin Metz
Martin MetzAffiliation: Klinik für Dermatologie, Charité, Berlin, Germany
Bio-protocol author page: a1176
Marcus Maurer
Marcus MaurerAffiliation: Klinik für Dermatologie, Charité, Berlin, Germany
Bio-protocol author page: a1177
 and Sandra M. O’Neill
Sandra M. O’NeillAffiliation: Biotechnology Department, Dublin City University, Dublin, Ireland
For correspondence: sandra.oneill@dcu.ie
Bio-protocol author page: a1178
Vol 4, Iss 4, 2/20/2014, 5486 views, 4 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1053

[Abstract] The generation of mast cells for in vitro studies comes from a variety of sources including mast cell lines (MC/9) (McCurdy et al., 2001), bone marrow-derived mast cells (BMMCs) (Supajatura et al., 2001), skin-derived mast cells (FSMCs) (Matsushima et al., 2004), peritoneal-derived mast cells (PMCs) (Hochdorfer et al., 2011) and peritoneal cell-derived cultured mast cells (PCMCs) (Vukman et al., 2012). BMMCs are generally used for in vitro studies because of the high yield of mast cells generated and also because they can be generated from knockout and transgenic mice making this a good source to examine specific factors important for mast cell function. Due to the large yield of cells generated they are the cells of choice for reconstitution studies in mast cell knockout mice (Sur et al., 2007). Furthermore, they are more responsive to both allergic and non-allergic stimuli when compared to mast cell lines. The major disadvantage of BMMCs is that they are not fully matured when compared to mast cells generated or obtained from other sources. For example, compared to PCMCs [see the protocol “Isolation and Culture of Peritoneal Cell-derived Mast Cells” (Vukman et al., 2014)], BMMCs express a restricted range of TLRs and cytokines when stimulated with TLR ligands (Mrabet-Dahbi et al., 2009). The different sources of mast cells can display phenotypical and functional differences and therefore it is important that when designing an experiment the correct cellular source is obtained. Here, we describe a protocol for the isolation and culture of murine mast cells from mouse bone marrow.

Keywords: Mast cells, Cell culture, Primary cells, Bone marrow, WEHI-3

Materials and Reagents

  1. C57BL/6 mice or mouse model of choice (Harlan Laboratories, catalog number: 057; Charles River Laboratories International, catalog number: BLCSIFE49D)
  2. Industrial methylated spirit (IMS) (Lennox Laboratory Supplies, catalog number: CRTS10330716)
  3. WEHI-3 conditioned medium generated from WEHI-3 cell line (ATCC, catalog number: TIB68)
  4. Sterile phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 14190)
  5. IL-3 (BioLegend, catalog number: 432101)
  6. IMDM with L-glutamine (Life Technologies, Gibco®, catalog number: 12440)
  7. Fetal calf serum (FCS) (Life Technologies, Gibco®, catalog number: 10270)
  8. Penicillin/streptomycin (Life Technologies, Gibco®, catalog number: 15140)
  9. Mercapto-ethanol (Sigma-Aldrich, catalog number: M3148)
  10. Trypan blue stain (Sigma-Aldrich, catalog number: T8154)
  11. Complete IMDM (see Recipes)
  12. Growth factors (see Recipes)
  13. Kimura dye (see Recipes)

Equipment

  1. Sterile forceps
  2. Sterile scissors
  3. Sterile pipette
  4. Syringe (10 ml)
  5. Needle (19-, 21- and 27-gauge)
  6. Falcon tube (50 ml)
  7. Cell scraper (SARSTEDT AG, catalog number: 83.1830)
  8. Filtropur S 0.45 filter (SARSTEDT AG, catalog number: 83.1826)
  9. Petri dishes (100 x 20 mm)
  10. PD100 petri dish
  11. Water bath
  12. Centrifuge
  13. T75 Cell culture flask (SARSTEDT AG, catalog number: 83.1813.502)
  14. T175 Cell culture flask (SARSTEDT AG, catalog number: 83.1812.502)
  15. Haemocytometer
  16. Safety cabinet

Procedure

Note: All procedures are performed in a sterile environment in a class II safety cabinet.

  1. Preparing WEHI-3 conditioned medium
    1. Thaw cells from stock (which is stored in liquid nitrogen) by incubating cells in a water bath at 37 °C for 2 min.
    2. Resuspend cells in 37 °C complete IMDM as quick as possible. Final cell number has to be 2 x 105 cells/ml and culture cells in T75 tissue culture flask at 37 °C, 5% CO2.
    3. Passages: Transfer medium from tissue culture flask into a 50 ml Falcon tube.
    4. Scrape cells using sterile cell scraper.
    5. Transfer scraped cells into the same 50 ml Falcon tube.
    6. Count cells, the concentration has to be > 6 x 105 cell/ml.
    7. Centrifuge cells at 300 x g for 5 min.
    8. Transfer supernatant into new Falcon tube.
    9. Resuspend cells in fresh complete IMDM (2 x 105 cell/ml).
    10. Plate cells into new flasks (T75/T175).
    11. Centrifuge the supernatant again at 600 x g for 15 min.
    12. Transfer and filter supernatant into new Falcon tube.
    13. Take 0.5 ml for measurement of IL-3 concentrations by commercial ELISA. The concentration should range between 10 to 100 ng/ml and a minimum of 10 ng/ml should be used.
    14. Freeze supernatants at -20 °C.
    15. WEHI-3 conditioned medium can be harvested every 3-4 days following the steps A3-14. The cell number should not be over 2 x 106.

  2. Isolation and culture of BMMCs
    Day 0 [Passage (P) 1]
    1. Kill mouse by cervical dislocation.
    2. Spray mouse thoroughly with 70% alcohol (or IMS) and lay down on 70% alcohol soaked paper.
    3. Two sets of sterile tweezers and scissors are used, one for the outer skin of the mouse and the other for the removal of legs (tibia and femur).
    4. Make a small incision below the sternum of the mouse and cut the skin away from the front around the back to make a complete circle. Peel the fur away completely from the lower half of the mouse leaving the legs exposed.
    5. Use the second pair of scissors and tweezers. Holding the tail with the index and middle finger and the leg of interest between the ring finger and thumb, cut the muscle away from the hind leg around the hip in a straight cutting manner so as to make the hip-joint amenable for cutting.
    6. Cut the hip-joint to free the leg from the body of the mouse. Be careful not to cut and hence expose the bone marrow of the femur. Repeat this with the second leg of the mouse.
    7. Cut the ankle of each leg to remove the feet, and discard them.
    8. Place legs in 50 ml tubes on ice partially filled with IMDM until next step.
    9. Place legs onto one half of a PD100 petri dish and remove residual muscle, fat and connective tissue using forceps and scissors.
    10. Cut the knee joint to separate the femur and tibia.
    11. Use fresh pair of forceps and scissors to cut each bone in turn at either end to expose the bone marrow.
    12. Flush the bone marrow onto the dish using a 10 ml syringe filled with ice cold media with a 27-gauge needle. Repeat this for all bones.
    13. Use a 19-gauge needle to break up and suspend the bone marrow by pipetting.
    14. Transfer the bone marrow suspension in to a 50 ml tube and centrifuged at 300 x g for 10 min.
    15. Discard the supernatant and resuspend cells in culture medium (IMDM with 30% WEHI-3 conditioned medium; 20 ml medium/mouse) and transfer suspension into tissue-treated cell culture flask (T75).
    16. Culture cells at 37 °C, 5% CO2.
    Day 5 (P2)
    1. Transfer cell suspension into a 50 ml tube (avoid taking adherent cells!). Pellet the cells by centrifugation (300 x g, 10 min, 4 °C), discard supernatant. Loosen pellet by flicking the tube and resuspend cells in 20 ml fresh culture medium and transfer cell suspension into a new tissue-treated cell culture flask (T75).
    Day 8 (P3)
    1. Repeat procedure as on day 5.
    Day 12 (P4)
    1. Transfer cell suspension into a new big tissue treated cell culture flask (T175), (avoid taking over adherent cells!) and add 20 ml fresh culture medium.
    Day 15 (P5), day 22 (P7), day 29 (P9)
    1. Same procedure as on day 5 but use a big (T175) culture flask.
    Day 19 (P6), day 26 (P8)
    1. Transfer 20 ml of cell suspension into a 50 ml tube. Pellet the cells by centrifugation (300 x g, 10 min, 4 °C). Discard supernatant. Loosen pellet by flicking the tube and resuspend cells in 20 ml fresh culture medium and transfer cell suspension back into the cell culture flask.
    2. When cells are 4 weeks of age (P9) count cells and determine purity of mast cells via Kimura stain or FACS-analysis. If the cells are > 95% pure, they can be used for experiments (Figure 1). If the purity is less, they have to be cultured another week.
      Note: Do not exceed a cell density of 2.5 x 106.


      Figure 1. The purity of bone marrow-derived cultured mast cells is over 95% after 4 weeks of cultivation. Bone marrow cells from C57BL/6 mouse were cultured for 4 weeks in IMDM (with 10% FCS, 100 U/ml penicillin/streptomycin and 50 μM 2-mercaptoethanol) in the presence of 30% WEHI-3 conditioned medium. Cell number (A) and purity (B) was determined by every passage, twice a week with trypan blue and Kimura staining. Data are presented as the mean ± SD of nine independent experiments.

Recipes

  1. Complete IMDM
    IMDM (500 ml)
    10% FCS (50 ml per 450 ml of IMDM)
    5 ml Penicillin (100 U/ml)/Streptomycin (100 μg/ml)
    1 M 2-mercaptoethanol (add 25 μl to 500 ml for 50 μM final concentration)
  2. Growth factors (add to IMDM prior to use)
    30% WEHI-3 conditioned medium
  3. Kimura dye
    Toluidine blue solution (50 ml) (0.5 mg/ml Toluidine blue, 18 g/l NaCl and 22% ethanol)
    Saturated saponin (2.27 ml) (4 mg/ml saponin in ethanol)
    NaH2PO4 solution (22.7 ml) (60 mM)

Acknowledgments

The protocol described here was adapted from Sur et al. (2007) and Vukman et al. (2012).

References

  1. Hochdorfer, T., Kuhny, M., Zorn, C. N., Hendriks, R. W., Vanhaesebroeck, B., Bohnacker, T., Krystal, G. and Huber, M. (2011). Activation of the PI3K pathway increases TLR-induced TNF-alpha and IL-6 but reduces IL-1beta production in mast cells. Cell Signal 23(5): 866-875.
  2. Matsushima, H., Yamada, N., Matsue, H. and Shimada, S. (2004). TLR3-, TLR7-, and TLR9-mediated production of proinflammatory cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells. J Immunol 173(1): 531-541.
  3. McCurdy, J. D., Lin, T. J. and Marshall, J. S. (2001). Toll-like receptor 4-mediated activation of murine mast cells. J Leukoc Biol 70(6): 977-984.
  4. Mrabet-Dahbi, S., Metz, M., Dudeck, A., Zuberbier, T. and Maurer, M. (2009). Murine mast cells secrete a unique profile of cytokines and prostaglandins in response to distinct TLR2 ligands. Exp Dermatol 18(5): 437-444.
  5. Supajatura, V., Ushio, H., Nakao, A., Okumura, K., Ra, C. and Ogawa, H. (2001). Protective roles of mast cells against enterobacterial infection are mediated by Toll-like receptor 4. J Immunol 167(4): 2250-2256.
  6. Sur, R., Cavender, D. and Malaviya, R. (2007). Different approaches to study mast cell functions. Int Immunopharmacol 7(5): 555-567.
  7. Vukman, K. V., Adams, P. N., Metz, M., Maurer, M. and O'Neill, S. M. (2013). Fasciola hepatica tegumental coat impairs mast cells' ability to drive Th1 immune responses. J Immunol 190(6): 2873-2879.
  8. Vukman, K. V., Metz, M., Maurer, M. and O'Neill, S. M. (2014). Isolation and culture of peritoneal cell-derived mast cells. Bio-protocol 4(4): e1052.
  9. Vukman, K. V., Visnovitz, T., Adams, P. N., Metz, M., Maurer, M. and O'Neill, S. M. (2012). Mast cells cultured from IL-3-treated mice show impaired responses to bacterial antigen stimulation. Inflamm Res 61(1): 79-85.


How to cite: Vukman, K. V., Metz, M., Maurer, M. and O’Neill, S. M. (2014). Isolation and Culture of Bone Marrow-derived Mast Cells. Bio-protocol 4(4): e1053. DOI: 10.21769/BioProtoc.1053; Full Text



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3/31/2015 5:57:49 AM  

Barbara Summers
Weill COrnell Medical College

Can you do a western on BMMC using anti FCεR1α to prove that your cells are mast cells or is FACS only possible with this antibody.

4/6/2015 11:25:12 PM  

Krisztina Vukman (Author)
Biotechnology Department,Dublin City University

You can do a western, but I think with that method you can only prove that you have mast cells in your cell culture, but are not able to check purity. I think Kimura stain is the easiest way to check purity. I ususally use facs, because it is very specific and reliable.

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3/25/2015 12:15:29 PM  

Barbara Summers
Weill COrnell Medical College

Why did you use Kimura staining? Is it specific for only mast cells?

3/30/2015 1:00:35 AM  

Krisztina Vukman (Author)
Biotechnology Department,Dublin City University

Kimura stain contains toluidine blue and it is often used to identify mast cells, because they stain heparin in their cytoplasmic granules. I tried it with some other bone marrow-derived cells and it was specific for mast cells. (Krisztina)

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11/21/2014 5:34:27 AM  

Barbara Summers
Weill COrnell Medical College

Our lab tried your protocol 3 times. Each time, 90% of the cells adhered to the flask. We had to hit the flask very hard to get cells in suspension. When replated, the cells stuck again. Any idea why we saw mostly adherent cells? After the first 5 days, will most of the cells be adherent?

11/25/2014 12:18:28 AM  

Sandra O’Neill (Author)
Biotechnology Department,Dublin City University

When feeding the cells it is important to take only the non-adherent cells. The cells adhered to the flask are not mast cells, so try not to get them in suspension. The mast cells yield in the begining is very low, but it will be 30-40 million by passage 9, and there will be less and less adherent cells. We also use flasks for non-adherent cells (cat.no. in the protocol).

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8/6/2014 8:43:34 AM  

Barbara Summers
Weill COrnell Medical College

Does it matter how old the mouse is? Will it work on a 5 month old mouse? How will the yield compare to a 4 week old mouse?

11/25/2014 12:06:03 AM  

Sandra O’Neill (Author)
Biotechnology Department,Dublin City University

We did not see any difference within BMMCs from old and young mice. In other labs they prefer to use old (few month old) mice.

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